CN111172077A - Microbial preparation for regulating live pig intestinal flora and preparation method thereof - Google Patents
Microbial preparation for regulating live pig intestinal flora and preparation method thereof Download PDFInfo
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- CN111172077A CN111172077A CN202010093360.6A CN202010093360A CN111172077A CN 111172077 A CN111172077 A CN 111172077A CN 202010093360 A CN202010093360 A CN 202010093360A CN 111172077 A CN111172077 A CN 111172077A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 71
- 230000000813 microbial effect Effects 0.000 title claims abstract description 59
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 41
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 83
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 62
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 62
- 241001134770 Bifidobacterium animalis Species 0.000 claims abstract description 52
- 229940118852 bifidobacterium animalis Drugs 0.000 claims abstract description 52
- 241000235646 Cyberlindnera jadinii Species 0.000 claims abstract description 32
- 240000001046 Lactobacillus acidophilus Species 0.000 claims abstract description 32
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims abstract description 32
- 244000199885 Lactobacillus bulgaricus Species 0.000 claims abstract description 32
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 claims abstract description 32
- 241000282887 Suidae Species 0.000 claims abstract description 32
- 229940039695 lactobacillus acidophilus Drugs 0.000 claims abstract description 32
- 229940004208 lactobacillus bulgaricus Drugs 0.000 claims abstract description 32
- 229940081969 saccharomyces cerevisiae Drugs 0.000 claims abstract description 32
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 31
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 31
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 31
- 239000002131 composite material Substances 0.000 claims abstract description 20
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 239000003674 animal food additive Substances 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 65
- 238000000855 fermentation Methods 0.000 claims description 54
- 230000004151 fermentation Effects 0.000 claims description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 49
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 241000186660 Lactobacillus Species 0.000 claims description 30
- 229940039696 lactobacillus Drugs 0.000 claims description 30
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 29
- 238000009630 liquid culture Methods 0.000 claims description 29
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 28
- 238000012258 culturing Methods 0.000 claims description 28
- 238000011081 inoculation Methods 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 23
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 20
- 235000013379 molasses Nutrition 0.000 claims description 18
- 239000002440 industrial waste Substances 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 17
- 239000001888 Peptone Substances 0.000 claims description 15
- 108010080698 Peptones Proteins 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 235000019319 peptone Nutrition 0.000 claims description 15
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 14
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 14
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 14
- 230000004913 activation Effects 0.000 claims description 13
- 239000002994 raw material Substances 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 12
- 230000001502 supplementing effect Effects 0.000 claims description 12
- 241000235342 Saccharomycetes Species 0.000 claims description 11
- 150000004676 glycans Chemical class 0.000 claims description 11
- 229920001282 polysaccharide Polymers 0.000 claims description 11
- 239000005017 polysaccharide Substances 0.000 claims description 11
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 11
- 240000008790 Musa x paradisiaca Species 0.000 claims description 10
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 10
- 235000019270 ammonium chloride Nutrition 0.000 claims description 10
- 239000011790 ferrous sulphate Substances 0.000 claims description 10
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 10
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 10
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 10
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 10
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 10
- 229960001763 zinc sulfate Drugs 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 9
- 241000186000 Bifidobacterium Species 0.000 claims description 7
- 150000003384 small molecules Chemical class 0.000 claims description 6
- 241000705930 Broussonetia papyrifera Species 0.000 claims description 5
- 244000269722 Thea sinensis Species 0.000 claims description 5
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 235000015099 wheat brans Nutrition 0.000 claims description 3
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 2
- 238000005507 spraying Methods 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims 1
- 230000012010 growth Effects 0.000 abstract description 13
- 244000005709 gut microbiome Species 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 35
- 241000894006 Bacteria Species 0.000 description 22
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 20
- 238000011068 loading method Methods 0.000 description 17
- 235000002639 sodium chloride Nutrition 0.000 description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 12
- 239000008103 glucose Substances 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 11
- 229920001817 Agar Polymers 0.000 description 10
- 239000008272 agar Substances 0.000 description 10
- 239000004310 lactic acid Substances 0.000 description 10
- 235000014655 lactic acid Nutrition 0.000 description 10
- 244000005700 microbiome Species 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- 229940041514 candida albicans extract Drugs 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 210000001035 gastrointestinal tract Anatomy 0.000 description 8
- 229920000136 polysorbate Polymers 0.000 description 8
- 239000012138 yeast extract Substances 0.000 description 8
- 230000009286 beneficial effect Effects 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 241000607142 Salmonella Species 0.000 description 5
- 235000014590 basal diet Nutrition 0.000 description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 4
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 240000003768 Solanum lycopersicum Species 0.000 description 4
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 229930003268 Vitamin C Natural products 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 235000015278 beef Nutrition 0.000 description 4
- 235000015190 carrot juice Nutrition 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 4
- 229940040511 liver extract Drugs 0.000 description 4
- 229940099596 manganese sulfate Drugs 0.000 description 4
- 239000011702 manganese sulphate Substances 0.000 description 4
- 235000007079 manganese sulphate Nutrition 0.000 description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 235000015193 tomato juice Nutrition 0.000 description 4
- 235000019154 vitamin C Nutrition 0.000 description 4
- 239000011718 vitamin C Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000000645 desinfectant Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- -1 diamine citrate Chemical class 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 210000003736 gastrointestinal content Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 2
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 229930182843 D-Lactic acid Natural products 0.000 description 1
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
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- 230000007366 host health Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
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- 238000011534 incubation Methods 0.000 description 1
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- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
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- 231100000252 nontoxic Toxicity 0.000 description 1
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- 235000006180 nutrition needs Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
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- 229920001184 polypeptide Polymers 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/33—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from molasses
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/123—Bulgaricus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Physiology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Birds (AREA)
- Sustainable Development (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of microbial feed additives, and particularly relates to a microbial preparation for regulating live pig intestinal flora and a preparation method thereof. The invention obtains the microbial preparation for regulating the intestinal flora of live pigs by sequentially carrying out activated culture, liquid strain culture, composite microbial preparation strain culture and finished product culture on lactobacillus acidophilus, lactobacillus bulgaricus, bifidobacterium animalis, saccharomyces cerevisiae, candida utilis, bacillus subtilis and bacillus licheniformis. The microbial preparation is further fermented with other components to prepare a pig fermented feed, and the pig fermented feed can be added into feed to change the composition of pig intestinal microbiota, adjust the intestinal microbial balance of live pigs and ensure the healthy growth of the live pigs.
Description
Technical Field
The invention belongs to the field of microbial feed additives, and particularly relates to a microbial preparation for regulating live pig intestinal flora and a preparation method thereof.
Background
The intestinal microbial ecosystem of animals is the biggest and most complex micro-ecosystem, and various bacteria are mutually restricted and interdependent. On one hand, the intestinal microorganisms can help the host digest food and provide various nutrients for the host; on the other hand, the micro-ecological balance in the intestinal tract of the host can be adjusted and maintained, and the micro-ecological balance has close relation with the growth, development, substance metabolism, aging and the like of the host, and plays an important role in the health of the host. The microbial preparation is a novel green feed additive developed in recent years, is non-toxic and residue-free, can regulate animal intestinal flora, inhibit harmful bacteria, promote growth and development, improve the ecological environment of livestock breeding, achieve the aim of ecological prevention and control, and has good economic benefit and ecological benefit.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a preparation method of a microbial preparation for regulating the live pig intestinal flora.
The invention also aims to provide the microbial preparation for regulating the live pig intestinal flora prepared by the preparation method.
The invention also aims to provide application of the microbial preparation for regulating the live pig intestinal flora.
The fourth purpose of the invention is to provide a pig fermentation feed, which is prepared by fermenting the microbial preparation for regulating the intestinal flora of the live pigs and other components.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a microbial preparation for regulating live pig intestinal flora comprises the following steps:
(1) and (3) strain activation culture: respectively activating freeze-preserved Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Bifidobacterium animalis (Bifidobacterium), saccharomyces cerevisiae (Saccharomyces cerevisiae), candida utilis (Candida utilis), Bacillus subtilis (Bacillus subtilis) and Bacillus licheniformis (Bacillus licheniformis) for multiple times to obtain activated strains of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium animalis, saccharomyces cerevisiae, candida utilis, Bacillus subtilis and Bacillus licheniformis;
(2) liquid strain culture
mixing and inoculating lactobacillus acidophilus and lactobacillus bulgaricus activated strains prepared in the step (1) in a liquid culture medium according to a mass ratio, and performing facultative anaerobic culture at 37 ℃ for 2-3 days to obtain a lactobacillus seed solution;
inoculating the bifidobacterium animalis activated strain prepared in the step (1) into a liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 2-3 days to obtain bifidobacterium animalis seed liquid;
③, mixing the saccharomyces cerevisiae and the candida utilis activated strain prepared in the step (1) in a mass ratio, inoculating the mixture into a liquid culture medium, and culturing for 2 days at the rotating speed of a shaking table of 150-200 r/min and the temperature of 28-30 ℃ to obtain a yeast seed solution;
mixing the bacillus subtilis and the bacillus licheniformis activated strain prepared in the step (1) in a mass ratio, inoculating the mixture into a liquid culture medium, and culturing for 1-2 days at a table concentrator rotating speed of 150-200 r/min and a temperature of 30-35 ℃ to obtain a bacillus seed solution;
(3) culturing a composite microbial preparation strain: inoculating the yeast seed liquid and the bacillus seed liquid prepared in the step (2) into a sterilized fermentation culture medium, culturing for 1-2 days at the temperature of 30 ℃ at the rotating speed of a shaking table of 150-200 r/min, then simultaneously inoculating the lactobacillus seed liquid and the bifidobacterium animalis seed liquid prepared in the step (2) into the fermentation system, and performing anaerobic culture for 3-5 days at the temperature of 37 ℃ to obtain a composite microbial preparation strain; wherein the mass ratio of the lactobacillus seed liquid to the bifidobacterium animalis seed liquid to the saccharomycete seed liquid to the bacillus seed liquid is 5:2:4:3, and the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation: inoculating 5-8% of the composite microbial preparation strain prepared in the step (3) into a fermentation culture medium according to the weight percentage, and performing closed fermentation at the temperature of 28-30 ℃ for 7-15 days to obtain a microbial preparation for regulating the intestinal flora of live pigs; wherein, the fermentation culture medium in the step is not sterilized;
the strains of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium animalis, saccharomyces cerevisiae, Candida utilis, Bacillus subtilis and Bacillus licheniformis in the step (1) are Lactobacillus acidophilus (Lactobacillus acidophilus) CICC 6086, Lactobacillus bulgaricus (Lactobacillus bulgaricus) ACCC 03958, Bifidobacterium animalis (Bifidobacterium animalis) CICC 6174, saccharomyces cerevisiae (saccharomyces cerevisiae) CICC 1355, Candida utilis (Candida utilis) CICC 31170, Bacillus subtilis (Bacillus subtilis) CICC 24434 and Bacillus licheniformis (Bacillus licheniformis) CGMCC1.10257 respectively;
the specific operation of the multiple activation in the step (1) is preferably:
respectively carrying out three times of slant activation on cryopreserved Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Bifidobacterium animalis (Bifidobacterium animalis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), candida utilis (Candida utilis), Bacillus subtilis (Bacillus subtilis) and Bacillus licheniformis (Bacillus licheniformis), then carrying out plate purification culture, and finally carrying out slant culture to obtain activated strains of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium animalis, Saccharomyces cerevisiae, candida utilis, Bacillus subtilis and Bacillus licheniformis;
the conditions of slant activation, plate purification culture or slant culture in step (1) are preferably:
carrying out facultative anaerobic culture on lactobacillus acidophilus or lactobacillus bulgaricus at 37 ℃ for 2-3 days;
carrying out anaerobic culture on the bifidobacterium animalis for 3-5 days at 37 ℃;
carrying out aerobic culture on saccharomyces cerevisiae or candida utilis at 28-30 ℃ for 2 days;
carrying out aerobic culture on bacillus subtilis or bacillus licheniformis for 2-3 days at 30 ℃;
the culture medium for the slant activation, plate purification culture or slant culture in the step (1) is preferably:
a culture medium for the culture of lactobacillus acidophilus or lactobacillus bulgaricus, comprising per liter the following components: 7.5 g of yeast extract, 7.5 g of peptone, 10 g of glucose, 2 g of monopotassium phosphate, 800.5 ml of tween, 20ml of tomato juice, 30ml of potato juice, 60ml of carrot juice, 2 g of vitamin C, 15 g of agar, supplementing 1000ml of weak alkaline small molecular reducing water, pH6.8, and sterilizing at 121 ℃ for 20 min;
a culture medium for the culture of bifidobacterium animalis comprising, per litre: 15.0 g of peptone, 2.0 g of yeast powder, 20.0 g of glucose, 0.5g of soluble starch, 5.0 g of sodium chloride, 10.0ml of 5% cysteine, 400.0ml of tomato extract, 801.0 ml of tween, 80.0ml of liver extract, 20.0 g of agar, supplementing 1000ml of weakly alkaline small molecular reducing water, pH7.0, and carrying out autoclaving at 115 ℃ for 15-20 min;
a culture medium for the cultivation of Saccharomyces cerevisiae or Candida utilis, comprising per liter the following components: 20 g of peptone, 10 g of yeast powder, 20 g of glucose, 20.0 g of agar and 1000ml of weakly alkaline small molecular reducing water, sterilizing at the temperature of 121 ℃ for 20min at the pH of 6.0;
a medium for culturing bacillus subtilis or bacillus licheniformis, each liter comprising the following components: 5g of yeast extract, 5g of beef extract, 10 g of peptone, 10 g of sodium chloride, 7.5 g of manganese sulfate, 0.5g of magnesium sulfate, 2 g of monopotassium phosphate, 15 g of agar and 1000ml of weakly alkaline small molecular reducing water, and sterilizing at the pH of 7.2 and the temperature of 121 ℃ for 20 min;
the liquid culture medium (lactic acid bacteria) in the step (2) ① contains the following components in each liter:
10 g of peptone, 10 g of beef extract, 5g of yeast powder, 5g of glucose, 5g of sodium acetate, 2 g of diamine citrate, 800.5 ml of tween, 2 g of dipotassium phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 20 g of calcium carbonate, 20ml of tomato juice, 30ml of potato juice, 60ml of carrot juice, 2 g of vitamin C and supplementing weakly alkaline small molecular reducing water to 1000ml, and sterilizing at the pH of 6.8 and 112 ℃ for 20 min;
step (2) and step (2), each liter of the liquid culture medium (bifidobacterium animalis) comprises the following components:
15.0 g of peptone, 2.0 g of yeast powder, 20.0 g of glucose, 0.5g of soluble starch, 5g of sodium chloride, 10.0ml of 5% cysteine, 400.0ml of tomato extract, 801.0 ml of tween, 80.0ml of liver extract, supplementing to 1000ml of weakly alkaline small molecular reducing water, and carrying out autoclaving at 115 ℃ for 15-20 min at the pH of 7.0;
step (2) the liquid culture medium (yeast) comprises the following components in each liter:
50 g of brown sugar, 20 g of yeast extract, 1 g of ammonium sulfate and 2 g of potassium chloride, wherein the pH is natural, the alkalescent micromolecule reducing water is supplemented to 1000ml, and the mixture is sterilized for 20min at the temperature of 121 ℃;
the liquid culture medium (bacillus) in the step (2) ④ contains the following components in each liter:
30 g of glucose, 15 g of yeast extract, 2 g of disodium hydrogen phosphate, 1 g of sodium dihydrogen phosphate and weakly alkaline small molecular reduced water, supplementing to 1000ml, sterilizing at the pH of 7.2 and the temperature of 112 ℃ for 20 min;
the fermentation medium in the step (3) comprises the following components in percentage by mass:
5% of industrial waste molasses, 0.5% of yeast powder, 1% of peptone, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of weakly alkaline small molecular reducing water which is supplemented to 100%;
the fermentation medium in the step (4) comprises the following components in percentage by mass:
5-8% of industrial waste molasses, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and a weakly alkaline small-molecule reducing water which is supplemented to 100%;
the specific operation of the closed fermentation in the step (4) is preferably:
firstly, cleaning culture containers and containers related to preparation of a culture medium, and disinfecting the containers by using acidic oxidation potential water (called acidified disinfectant water for short);
weighing the components (banana polysaccharide, ammonium chloride, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate and the like) of the fermentation medium according to the proportion, adding the components into the container 1, and then adding weak alkaline small molecule reducing water to fully dissolve the components;
thirdly, weighing industrial waste molasses according to the proportion, adding the industrial waste molasses into the container 2, and then adding weak alkaline small molecule reducing water to fully dissolve the industrial waste molasses;
adding the solution prepared in the step ② and the solution prepared in the step ② into a culture container, and then adding weak alkaline small molecule reduction water to 80% of the total volume of the culture medium;
fifthly, after shaking up the strains of the compound microbial preparation prepared in the step (3), inoculating the strains into a culture container according to the inoculation amount (relative to the total amount of the culture medium) of 5-8% by weight;
sixthly, after inoculation, adding weak alkaline micromolecule reducing water into a culture container until the total volume of the culture medium is 100%, uniformly stirring, and carrying out closed fermentation at the temperature of 28-30 ℃ for 7-15 days to obtain a microbial preparation for regulating the intestinal flora of the live pigs;
a microbial preparation for regulating live pig intestinal flora is prepared by the above preparation method;
the microbial preparation for regulating the live pig intestinal flora is applied to the preparation of a pig feed additive;
a pig fermentation material is prepared by fermenting the following raw materials in parts by mass:
the broussonetia papyrifera leaves and the tea leaves are preferably dried broussonetia papyrifera leaves and tea leaves;
the preparation method of the pig fermented feed comprises the following steps:
respectively crushing, screening and uniformly mixing corn, bean pulp, wheat bran, broussonetia papyrifera leaves and tea leaves to obtain a mixed raw material; uniformly mixing a microbial preparation for regulating the intestinal flora of live pigs and industrial waste molasses with 50 parts by mass of water, and spraying the mixture onto mixed raw materials; carrying out sealed fermentation, wherein the fermentation is carried out for 10-15 days in summer and for 15-20 days in winter, so as to obtain the pig fermentation material;
the screening mesh number is preferably 40 meshes;
the application of the pig fermented feed in preparing pig feed;
the pig feed preferably contains 5-15 wt% of pig fermentation material;
compared with the prior art, the invention has the following advantages and effects:
(1) the invention adopts mixed culture technology, and various microorganisms mutually provide specific growth nutrients; improving the biomass and physiological metabolism function of the microorganism; makes up the deficiency of single culture metabolism, promotes the growth of compound microorganisms by symbiosis, paragenetic symbiosis and the like, and has a plurality of obvious advantages compared with the traditional pure culture technology.
(2) The fermentation medium is directly used for culturing the compound microbial preparation without being disinfected in the finished product fermentation process, and is also not required to be stirred in the culture process, so that manpower and material resources can be saved, and the economic benefit is improved.
(3) The active ingredients of the microbial preparation for regulating the live pig intestinal flora mainly comprise live bacteria, dead bacteria and metabolites, and the microbial preparation is mainly used for changing the composition of the intestinal microbiota, enabling beneficial or harmless microorganisms to occupy population advantages, and regulating the live pig intestinal microbial ecological balance by competitively inhibiting the proliferation of pathogenic bacteria or harmful microorganisms. After the beneficial microorganisms enter the live pig intestinal tract, the beneficial microorganisms can rapidly form dominant microbial flora together with beneficial bacteria in the intestinal tract to inhibit the growth of harmful bacteria in the intestinal tract, wherein the beneficial microorganisms are tightly combined with intestinal mucosa epithelial cells through teichoic acid around the intestinal tract wall to form a bacterial membrane barrier, prevent the colonization of harmful bacteria and regulate the microecological balance of the live pig intestinal tract.
(4) The invention utilizes the prepared microbial preparation for regulating the intestinal flora of the live pigs to ferment the feed raw materials, and the growth and metabolism of the saccharomycetes are taken as the main raw materials in the initial fermentation stage, and the saccharomycetes can quickly consume O2And can endure strong acidic environment (about pH 3.2). When the yeast is consumed O2Later, favorable conditions are created for the growth and the propagation of bacillus, lactic acid bacteria and animal bifidobacteria. With the growth and metabolism of the lactic acid bacteria, the number of the lactic acid bacteria is continuously increased, some macromolecular nutrient substances (such as protein, polysaccharide, fat and the like) are continuously converted into micromolecular nutrient substances, organic acid, polypeptide, free amino acid, vitamin and the like are continuously increased, and the nutritive value of the feed is greatly improved. During fermentation, lactobacillus and bifidobacterium can produce organic acid such as lactic acid, acetic acid and the like, the pH value is reduced, the proliferation of escherichia coli, salmonella and the like is inhibited, the bacillus produces antibacterial peptide, and the lactobacillus produces bacteriocin, which can effectively kill harmful bacteria (such as escherichia coli and salmonella) in feed raw materials. The fermentation is close to the mature period, the lactic acid bacteria are absolutely dominant, the quantity of animal bifidobacterium strains, yeasts and bacilli tends to be stable, and a complex and stable microecosystem with multiple functions is formed. The production of the fermented feed can be completed only by three processes of inoculation, mixing and fermentation. The raw materials can be directly used for fermentation production without being sterilized, manual turning is not needed in the production process, the fermentation finished product does not need to be dried, a large amount of labor force can be saved, and the labor intensity of workers is reduced.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
The strains of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium animalis, saccharomyces cerevisiae, Candida utilis, bacillus subtilis and bacillus licheniformis referred to in the examples are Lactobacillus acidophilus (Lactobacillus acidophilus) cic 6086, Lactobacillus bulgaricus (Lactobacillus bulgaricus) acc 03958, Bifidobacterium animalis (Bifidobacterium animalis) cic 6174, saccharomyces cerevisiae (saccharomyces cerevisiae) cic 1355, Candida utilis (Candida utilis) cic 31170, respectively; bacillus subtilis CICC 24434 and Bacillus licheniformis CGMCC 1.10257;
the acidic electrolyzed oxidizing water (called acidified disinfectant water for short) in the embodiment is an aqueous solution which is prepared by adding less than 0.1 wt% of salt (sodium chloride) into water, inputting the aqueous solution into an electrolytic cell for electrolysis, generating an anode of the electrolytic cell, wherein the pH value of the aqueous solution is 2-3, the oxidation-reduction potential of the aqueous solution is more than 1100mV, and the active chlorine is 50-70 mg.
In the examples, during the strain activation culture (slant activation, plate purification culture or slant culture), the culture medium of each strain was:
a culture medium for the culture of lactobacillus acidophilus or lactobacillus bulgaricus, comprising per liter the following components: 7.5 g of yeast extract, 7.5 g of peptone, 10 g of glucose, 2 g of monopotassium phosphate, 800.5 ml of tween, 20ml of tomato juice, 30ml of potato juice, 60ml of carrot juice, 2 g of vitamin C, 15 g of agar, supplementing 1000ml of weak alkaline small molecular reducing water, pH6.8, and sterilizing at 121 ℃ for 20 min;
a culture medium for the culture of bifidobacterium animalis comprising, per litre: 15.0 g of peptone, 2.0 g of yeast powder, 20.0 g of glucose, 0.5g of soluble starch, 5.0 g of sodium chloride, 10.0ml of 5% cysteine, 400.0ml of tomato extract, 801.0 ml of tween, 80.0ml of liver extract, 20.0 g of agar, supplementing 1000ml of weakly alkaline small molecular reducing water, pH7.0, and carrying out autoclaving at 115 ℃ for 15-20 min;
a culture medium for the cultivation of Saccharomyces cerevisiae or Candida utilis, comprising per liter the following components: 20 g of peptone, 10 g of yeast powder, 20 g of glucose, 20.0 g of agar and 1000ml of weakly alkaline small molecular reducing water, sterilizing at the temperature of 121 ℃ for 20min at the pH of 6.0;
a medium for culturing bacillus subtilis or bacillus licheniformis, each liter comprising the following components: 5g of yeast extract, 5g of beef extract, 10 g of peptone, 10 g of sodium chloride, 7.5 g of manganese sulfate, 0.5g of magnesium sulfate, 2 g of monopotassium phosphate, 15 g of agar and 1000ml of weakly alkaline small molecular reducing water, and sterilizing at the pH of 7.2 and the temperature of 121 ℃ for 20 min;
in the liquid strain culture process in the examples, the culture medium of each strain is:
the liquid culture medium (lactic acid bacteria) in the step (2) ① contains the following components in each liter:
10 g of peptone, 10 g of beef extract, 5g of yeast powder, 5g of glucose, 5g of sodium acetate, 2 g of diamine citrate, 800.5 ml of tween, 2 g of dipotassium phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 20 g of calcium carbonate, 20ml of tomato juice, 30ml of potato juice, 60ml of carrot juice, 2 g of vitamin C and supplementing weakly alkaline small molecular reducing water to 1000ml, and sterilizing at the pH of 6.8 and 112 ℃ for 20 min;
step (2) and step (2), each liter of the liquid culture medium (bifidobacterium animalis) comprises the following components:
15.0 g of peptone, 2.0 g of yeast powder, 20.0 g of glucose, 0.5g of soluble starch, 5g of sodium chloride, 10.0ml of 5% cysteine, 400.0ml of tomato extract, 801.0 ml of tween, 80.0ml of liver extract, supplementing to 1000ml of weakly alkaline small molecular reducing water, and carrying out autoclaving at 115 ℃ for 15-20 min at the pH of 7.0;
step (2) the liquid culture medium (yeast) comprises the following components in each liter:
50 g of brown sugar, 20 g of yeast extract, 1 g of ammonium sulfate, 2 g of potassium chloride, natural pH, supplementing 1000ml of weakly alkaline micromolecular reducing water, and sterilizing for 20min at 121 ℃;
the liquid culture medium (bacillus) in the step (2) ④ contains the following components in each liter:
30 g of glucose, 15 g of yeast extract, 2 g of disodium hydrogen phosphate, 1 g of sodium dihydrogen phosphate and weakly alkaline small molecular reduced water, supplementing to 1000ml, sterilizing at the pH of 7.2 and the temperature of 112 ℃ for 20 min;
in the culture process of the composite microbial preparation strain in the embodiment, the fermentation medium comprises the following components in percentage by mass:
5% of industrial waste molasses, 0.5% of yeast powder, 1% of peptone, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of weakly alkaline small molecular reducing water which is supplemented to 100%;
example 1
(1) And (3) strain activation culture: respectively activating lactobacillus acidophilus, lactobacillus bulgaricus, bifidobacterium animalis, saccharomyces cerevisiae, candida utilis, bacillus subtilis and bacillus licheniformis in a frozen storage tube for three times in an inclined plane of a test tube at 18x180mm, then respectively placing the activated bacteria in a culture dish with 90mm for purification culture, and then respectively culturing the bacteria in an inclined plane of a test tube at 18x180mm to obtain activated bacteria of the lactobacillus acidophilus, the lactobacillus bulgaricus, the bifidobacterium animalis, the saccharomyces cerevisiae, the candida utilis, the bacillus subtilis and the bacillus licheniformis; wherein, the culture conditions are as follows: lactobacillus acidophilus and Lactobacillus bulgaricus are subjected to facultative anaerobic culture at 37 ℃ for 2 days, bifidobacterium animalis is subjected to anaerobic culture at 37 ℃ for 5 days, Saccharomyces cerevisiae and Candida utilis are subjected to aerobic culture at 28 ℃ for 2 days, and Bacillus subtilis and Bacillus licheniformis are subjected to aerobic culture at 30 ℃ for 2 days.
(2) Liquid strain culture
firstly, mixing and inoculating lactobacillus acidophilus and lactobacillus bulgaricus activated strains in a sterilized liquid culture medium according to a mass ratio, wherein the liquid loading amount is 90mL/100mL, and performing facultative anaerobic culture at 37 ℃ for 2 days to obtain a lactobacillus seed solution;
inoculating the bifidobacterium animalis activated strain into a sterilized liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 2 days to obtain a bifidobacterium animalis seed solution, wherein the liquid loading amount is 90mL/100 mL;
thirdly, mixing and inoculating saccharomyces cerevisiae and candida utilis activated strains in a sterilized liquid culture medium according to the mass ratio, culturing for 2 days at the rotating speed of a shaker of 150r/min and the temperature of 30 ℃ and the liquid loading amount of 25mL/250mL to obtain a yeast seed solution;
mixing and inoculating bacillus subtilis and bacillus licheniformis activated strains in a sterilized liquid culture medium according to the mass ratio, and culturing for 1 day at the rotating speed of a shaker of 200r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL to obtain a bacillus seed solution;
(3) culturing a composite strain: inoculating the yeast seed liquid and the bacillus seed liquid prepared in the step (2) into a sterilized fermentation culture medium, culturing for 1 day at the rotating speed of a shaking table of 200r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, then simultaneously inoculating the lactobacillus seed liquid and the animal bifidobacterium seed liquid prepared in the step (2) into the fermentation system, and performing anaerobic culture for 5 days at the temperature of 37 ℃ to obtain a compound microbial preparation strain; wherein the mass ratio of the lactobacillus seed liquid to the bifidobacterium animalis seed liquid to the saccharomycete seed liquid to the bacillus seed liquid is 5:2:4:3, and the total inoculation amount is 10 percent by weight;
(4) culture of finished products
firstly, cleaning culture containers and containers related to preparation of a culture medium, and disinfecting the containers by using acidic oxidation potential water (called acidified disinfectant water for short);
②, weighing banana polysaccharide, ammonium chloride, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate and the like which are components of the fermentation medium according to the proportion, adding the components into the container 1, and then adding weak alkaline micromolecule reducing water to fully dissolve the components;
thirdly, weighing industrial waste molasses according to the proportion, adding the industrial waste molasses into the container 2, and then adding weak alkaline small molecule reducing water to fully dissolve the industrial waste molasses;
adding the solution prepared in the step ② and the solution prepared in the step ② into a culture container, washing a container for dissolving the culture medium and molasses with a small amount of weak alkaline micromolecule reducing water for several times to ensure that all the components are added into the culture container, and then adding the weak alkaline micromolecule reducing water to 80% of the total volume of the culture medium;
fifthly, shaking the composite microbial preparation strain prepared in the step (3) uniformly, inoculating the strain into a culture container according to the inoculation amount (relative to the total amount of the culture medium) of 5 percent by weight, measuring a small amount of weak alkaline micromolecule reduced water in the strain container, and washing the strain container for several times to ensure that the strain is completely added into the culture container;
sixthly, after inoculation, adding weak alkaline micromolecule reducing water into a culture container until the total volume of the culture medium is 100%, uniformly stirring, covering a cover, and hermetically fermenting for 15 days at 28 ℃ to obtain the microbial preparation for regulating the intestinal flora of the live pigs, wherein the fermentation culture medium comprises the following components, by mass, 5% of industrial waste molasses, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate, and the balance of weak alkaline micromolecule reducing water being 100%.
Example 2
(1) And (3) strain activation culture: the specific method is the same as example 1, wherein the culture conditions are specifically as follows: facultative anaerobic culture of acidophilic lactobacillus and Bulgaria lactobacillus at 37 deg.c for 3 days, anaerobic culture of animal bifidobacterium at 37 deg.c for 4 days, aerobic culture of Saccharomyces cerevisiae and Candida utilis at 30 deg.c for 2 days, and aerobic culture of Bacillus subtilis and Bacillus licheniformis at 30 deg.c for 2 days;
(2) liquid strain culture
firstly, mixing and inoculating lactobacillus acidophilus and lactobacillus bulgaricus activated strains in a sterilized liquid culture medium according to a mass ratio, wherein the liquid loading amount is 90mL/100mL, and performing facultative anaerobic culture at 37 ℃ for 2.5 days to obtain a lactobacillus seed solution;
inoculating the bifidobacterium animalis activated strain into a sterilized liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 3 days to obtain a bifidobacterium animalis seed solution, wherein the liquid loading amount is 90mL/100 mL;
thirdly, mixing and inoculating saccharomyces cerevisiae and candida utilis activated strains in a sterilized liquid culture medium according to the mass ratio, culturing for 2 days at the rotating speed of a shaking table of 180r/min and the temperature of 29 ℃ and the liquid loading amount of 25mL/250mL to obtain a yeast seed solution;
fourthly, mixing and inoculating the bacillus subtilis and the bacillus licheniformis activated strains in the sterilized liquid culture medium according to the mass ratio, culturing for 1.5 days at the rotating speed of 180r/min and the temperature of 32 ℃ and the liquid loading amount of 30mL/250mL of a shaking table to obtain a bacillus seed solution;
(3) culturing a composite strain: inoculating the yeast seed liquid and the bacillus seed liquid prepared in the step (2) into a sterilized fermentation culture medium, culturing for 2 days at the rotating speed of a shaking table of 180r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, then simultaneously inoculating the lactobacillus seed liquid and the bifidobacterium animalis seed liquid prepared in the step (2) into the fermentation system, and performing anaerobic culture for 3 days at the temperature of 37 ℃ to obtain a compound microbial preparation strain; wherein the mass ratio of the lactobacillus seed liquid to the bifidobacterium animalis seed liquid to the saccharomycete seed liquid to the bacillus seed liquid is 5:2:4:3, and the total inoculation amount is 10 percent by weight;
(4) culture of finished products
the steps of (1) to (④) are the same as in example 1;
fifthly, shaking the composite microbial preparation strain prepared in the step (3) uniformly, inoculating the strain into a culture container according to the inoculation amount (relative to the total amount of the culture medium) of 6 percent by weight, measuring a small amount of weak alkaline micromolecule reduced water in the strain container, and washing the strain container for several times to ensure that the strain is completely added into the culture container;
sixthly, after inoculation, adding weak alkaline micromolecule reducing water into a culture container until the total volume of the culture medium is 100%, uniformly stirring, covering a cover, and hermetically fermenting at 29 ℃ for 10 days to obtain the microbial preparation for regulating the intestinal flora of the live pigs, wherein the fermentation culture medium comprises the following components, by mass, 6% of industrial waste molasses, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate, and the balance of weak alkaline micromolecule reducing water being 100%.
Example 3
(1) And (3) strain activation culture: the specific method is the same as example 1, wherein the culture conditions are specifically as follows: lactobacillus acidophilus and Lactobacillus bulgaricus are subjected to facultative anaerobic culture at 37 ℃ for 3 days, bifidobacterium animalis is subjected to anaerobic culture at 37 ℃ for 3 days, Saccharomyces cerevisiae and Candida utilis are subjected to aerobic culture at 29 ℃ for 2 days, and Bacillus subtilis and Bacillus licheniformis are subjected to aerobic culture at 30 ℃ for 3 days.
(2) Liquid strain culture
firstly, mixing and inoculating lactobacillus acidophilus and lactobacillus bulgaricus activated strains in a sterilized liquid culture medium according to a mass ratio, wherein the liquid loading amount is 90mL/100mL, and performing facultative anaerobic culture at 37 ℃ for 3 days to obtain a lactobacillus seed solution;
inoculating the bifidobacterium animalis activated strain into a sterilized liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 2.5 days to obtain a bifidobacterium animalis seed solution, wherein the liquid loading amount is 90mL/100 mL;
thirdly, mixing and inoculating saccharomyces cerevisiae and candida utilis activated strains in a sterilized liquid culture medium according to the mass ratio, culturing for 2 days at the rotating speed of a shaker of 200r/min and the temperature of 28 ℃ and the liquid loading amount of 25mL/250mL to obtain a yeast seed solution;
mixing and inoculating bacillus subtilis and bacillus licheniformis activated strains in a sterilized liquid culture medium according to the mass ratio, culturing for 1 day at the rotating speed of a shaker of 150r/min and the temperature of 35 ℃ and the liquid loading amount of 30mL/250mL to obtain a bacillus seed solution;
(3) culturing a composite strain: inoculating the yeast seed liquid and the bacillus seed liquid prepared in the step (2) into a sterilized fermentation culture medium, culturing for 1 day at the rotating speed of a shaking table of 150r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, then simultaneously inoculating the lactobacillus seed liquid and the bifidobacterium animalis seed liquid prepared in the step (2) into the fermentation system, and performing anaerobic culture for 4 days at 37 ℃ to obtain a composite microbial preparation strain; wherein the mass ratio of the lactobacillus seed liquid to the bifidobacterium animalis seed liquid to the saccharomycete seed liquid to the bacillus seed liquid is 5:2:4:3, and the total inoculation amount is 10 percent by weight;
(4) culture of finished products
the steps of (1) to (④) are the same as in example 1;
⑤, shaking the strains of the compound microbial preparation prepared in the step (3), inoculating the strains into a culture container according to the inoculation amount (relative to the total amount of a culture medium) of 8 percent of the weight percentage, and measuring a small amount of weak alkaline micromolecule reduced water in the strain container to wash the strain container for several times so as to ensure that the strains are all added into the culture container;
sixthly, after inoculation, adding weak alkaline micromolecule reducing water into a culture container until the total volume of the culture medium is 100%, uniformly stirring, covering a cover, and hermetically fermenting for 7 days at 30 ℃ to obtain the microbial preparation for regulating the intestinal flora of the live pigs, wherein the fermentation culture medium comprises 8 mass percent of industrial waste molasses, 0.3 mass percent of banana polysaccharide, 0.2 mass percent of ammonium chloride, 0.05 mass percent of sodium chloride, 0.1 mass percent of potassium dihydrogen phosphate, 0.05 mass percent of magnesium sulfate, 0.025 mass percent of zinc sulfate, 0.025 mass percent of ferrous sulfate, and the balance of weak alkaline micromolecule reducing water is 100%.
Comparative example 1
(1) And (3) strain activation culture: the concrete method is the same as the embodiment 2;
(2) liquid strain culture: the concrete method is the same as the embodiment 2;
(3) culturing a composite strain: simultaneously inoculating the lactobacillus seed liquid, the bifidobacterium animalis seed liquid, the saccharomycete seed liquid and the bacillus seed liquid prepared in the step (2) into a sterilized fermentation culture medium, culturing for 2 days at the rotating speed of a shaker of 180r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, and then performing anaerobic culture for 3 days at the temperature of 37 ℃ to obtain a composite microbial preparation strain; wherein the mass ratio of the lactobacillus seed liquid to the bifidobacterium animalis seed liquid to the saccharomycete seed liquid to the bacillus seed liquid is 5:2:4:3, and the total inoculation amount is 10 percent by weight.
Comparative example 2
(1) And (3) strain activation culture: the concrete method is the same as the embodiment 2;
(2) liquid strain culture: the concrete method is the same as the embodiment 2;
(3) culturing a composite strain: inoculating the lactobacillus seed solution and the bifidobacterium animalis seed solution prepared in the step (2) into a sterilized fermentation culture medium, and carrying out anaerobic culture at 37 ℃ for 3 days; then inoculating the yeast seed liquid and the bacillus seed liquid prepared in the step (2) into the fermentation system at the same time, and culturing for 2 days at the rotating speed of a shaking table of 180r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL to obtain a composite microbial preparation strain; wherein the mass ratio of the lactobacillus seed liquid to the bifidobacterium animalis seed liquid to the saccharomycete seed liquid to the bacillus seed liquid is 5:2:4:3, and the total inoculation amount is 10 percent by weight.
Effect example 1
Comparative example 1 lactic acid bacteria seed solution, bifidobacterium animalis seed solution, yeast seed solution and bacillus seed solution were simultaneously inoculated into a sterilized fermentation medium, and aerobic culture was performed for 2 days, followed by anaerobic culture for 3 days; comparative example 2 is that the lactobacillus seed solution and the bifidobacterium animalis seed solution are inoculated in the fermentation medium and anaerobically cultured for 3 days, and then the saccharomycete seed solution and the bacillus seed solution are inoculated and aerobically cultured for 2 days; example 2 inoculating a yeast seed solution and a bacillus seed solution into a fermentation culture medium, carrying out aerobic culture for 2 days, then inoculating a lactobacillus seed solution and a bifidobacterium animalis seed solution, and carrying out anaerobic culture for 3 days; the other parameters are the same.
Table 1 shows the results of counting each strain in the composite microbial preparation strains obtained in example 2 and comparative examples 1 and 2, and it can be seen from Table 1 that the Bacillus species died largely in the late stage of culture in the order of inoculation in comparative example 1; according to the inoculation sequence of comparative example 2, bacillus and yeast did not grow well; the higher growth was achieved for the cells according to the inoculation sequence of example 2. This indicates that the inoculation sequence of example 2 is the optimal inoculation sequence.
TABLE 1 results of different inoculation sequence counts ((cfu/ml))
| Inoculation sequence | Lactic acid bacteria | Bifidobacterium animalis | Bacillus | Yeast |
| Comparative example 1 | 1.43×109 | 2.23×108 | 1.13×105 | 6.40×106 |
| Comparative example 2 | 1.51×109 | 3.67×18 | 1.35×105 | 6.28×106 |
| Example 2 | 1.97×1010 | 8.41×109 | 6.31×108 | 7.92×109 |
Application examples
(1) Respectively crushing 60kg of corn, 25kg of soybean meal, 5kg of wheat bran, 5kg of broussonetia papyrifera leaves (dried in the sun) and 5kg of tea leaves (dried in the sun), sieving by a 40-mesh sieve, and uniformly mixing to obtain a mixed raw material;
(2) 3kg of the microbial preparation for regulating the intestinal flora of the live pigs, which is prepared in the example 2, 1kg of industrial waste molasses and 50kg of water are uniformly mixed, then the mixture is sprayed on the mixed raw materials, the raw materials are stirred while being sprayed, the mixture is uniformly mixed, the moisture is kneaded into a mass by hand, and the mass is not dropped, and the mass is preferably dispersed by touching; then placing into a thick plastic bag, tightening with a binding belt, fermenting at room temperature in a rat-proof place for 15 days until the fermentation is finished with wine flavor, and obtaining the fermented feed for pigs.
Effect example 2
In order to better illustrate that the pig fermentation feed prepared by the application example of the invention can promote the colonization of beneficial microorganisms in the intestinal tract of live pigs, further influence the intestinal flora environment and adjust the intestinal microecological balance. The inventors conducted the following tests. Wherein, the lactic acid bacteria liquid in the embodiment is not less than 108CFU/ml) is obtained by mixing Lactobacillus acidophilus and Lactobacillus bulgaricus activated strains in a mass ratio, inoculating into a sterilized liquid culture medium, and performing facultative anaerobic culture at 37 ℃ for 3 days.
The test is carried out in the eight-ring kingdom of Anyuan county of Ganzhou city, Jiangxi province, and 300 Du, Dada and Chang hybrid pigs with the weight of about 30kg are selected as the live pigs for the test and randomly divided into 5 groups. Each set of 3 replicates, each 20 replicates. Feeding 5 different treatment diets: basal diet (control group), basal diet +5 wt% of pig fermented material (treatment 1), basal diet +10 wt% of pig fermented material (treatment 2), basal diet + 5% of lactobacillus bacterial solution (treatment 3), basal diet + 10% of lactobacillus bacterial solution (treatment 4). The basic ration is prepared according to the nutrition needs of growing pigs, and the test period is 70 days. Feeding powder, manual feeding and free feeding. It is suitable for people to eat food slightly left, and is fed for 3 times daily, and the nipple type drinking device can drink water freely. The immunization procedure was performed as usual.
At the end of the test, randomly selecting 2 pigs every time, taking 0.5g of intestinal content from the rectum into a 7mL sterile centrifuge tube, adding 4.5mL of sterile normal saline, fully and uniformly mixing the diluent by using a vortex mixer, and sucking 100uL of supernatant into 18x180mm test tubes containing 900uL of sterile normal saline to sequentially dilute by 10 times. The bacterial count was determined by plate colony counting after 48h incubation at 37 ℃ and expressed as CFU/g bacteria per g of intestinal contents. The Escherichia coli culture uses Macconka agar medium, the Salmonella culture uses SS agar medium, and the lactic acid bacteria culture uses MRS medium.
The test results are shown in table 2, compared with the control group, the intestinal escherichia coli numbers of the pigs treated by treatment 1, treatment 2, treatment 3 and treatment 4 are respectively reduced by 35.96%, 41.77%, 11.62% and 15.35%, the salmonella numbers are respectively reduced by 44.30%, 51.26%, 14.37% and 17.19% compared with the control group, the intestinal lactobacillus numbers of the test groups are all higher than that of the control group, and are respectively improved by 240.74%, 284.20%, 182.79% and 184.86% compared with the control group, but the treatment 1 and the treatment 2 are obviously better than the treatment 3 and the treatment 4.
TABLE 2 Effect of Complex microbial preparation on porcine intestinal flora (CFU/g)
Therefore, when the live pigs enter the growth period, the intestinal flora gradually changes, the number of pathogenic bacteria such as escherichia coli and the like rises, and the number of beneficial bacteria such as lactobacillus and the like falls; the composite microbial preparation fermentation material added into the daily ration can improve the growth performance of the live pigs, reduce the quantity of escherichia coli and salmonella in intestinal tracts of the live pigs, promote the proliferation of intestinal lactobacillus and ensure the healthy growth of the live pigs.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of a microbial preparation for regulating live pig intestinal flora is characterized by comprising the following steps:
(1) and (3) strain activation culture: respectively activating freeze-preserved Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Bifidobacterium animalis (Bifidobacterium), saccharomyces cerevisiae (Saccharomyces cerevisiae), candida utilis (Candida utilis), Bacillus subtilis (Bacillus subtilis) and Bacillus licheniformis (Bacillus licheniformis) for multiple times to obtain activated strains of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium animalis, saccharomyces cerevisiae, candida utilis, Bacillus subtilis and Bacillus licheniformis;
(2) liquid strain culture
mixing and inoculating lactobacillus acidophilus and lactobacillus bulgaricus activated strains prepared in the step (1) in a liquid culture medium according to a mass ratio, and performing facultative anaerobic culture at 37 ℃ for 2-3 days to obtain a lactobacillus seed solution;
inoculating the bifidobacterium animalis activated strain prepared in the step (1) into a liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 2-3 days to obtain bifidobacterium animalis seed liquid;
③, mixing the saccharomyces cerevisiae and the candida utilis activated strain prepared in the step (1) in a mass ratio, inoculating the mixture into a liquid culture medium, and culturing for 2 days at the rotating speed of a shaking table of 150-200 r/min and the temperature of 28-30 ℃ to obtain a yeast seed solution;
mixing the bacillus subtilis and the bacillus licheniformis activated strain prepared in the step (1) in a mass ratio, inoculating the mixture into a liquid culture medium, and culturing for 1-2 days at a table concentrator rotating speed of 150-200 r/min and a temperature of 30-35 ℃ to obtain a bacillus seed solution;
(3) culturing a composite microbial preparation strain: inoculating the yeast seed liquid and the bacillus seed liquid prepared in the step (2) into a sterilized fermentation culture medium, culturing for 1-2 days at the temperature of 30 ℃ at the rotating speed of a shaking table of 150-200 r/min, then simultaneously inoculating the lactobacillus seed liquid and the bifidobacterium animalis seed liquid prepared in the step (2) into the fermentation system, and performing anaerobic culture for 3-5 days at the temperature of 37 ℃ to obtain a composite microbial preparation strain; wherein the mass ratio of the lactobacillus seed liquid to the bifidobacterium animalis seed liquid to the saccharomycete seed liquid to the bacillus seed liquid is 5:2:4:3, and the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation: inoculating 5-8% of the composite microbial preparation strain prepared in the step (3) into a fermentation culture medium according to the weight percentage, and performing closed fermentation at the temperature of 28-30 ℃ for 7-15 days to obtain a microbial preparation for regulating the intestinal flora of live pigs; wherein the fermentation medium is not sterilized in this step.
2. The method for preparing a microbial preparation for regulating the intestinal flora of live pigs according to claim 1, wherein the preparation method comprises the following steps:
the strains of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium animalis, saccharomyces cerevisiae, Candida utilis, Bacillus subtilis and Bacillus licheniformis in the step (1) are Lactobacillus acidophilus (Lactobacillus acidophilus) CICC 6086, Lactobacillus bulgaricus (Lactobacillus bulgaricus) ACCC 03958, Bifidobacterium animalis (Bifidobacterium animalis) CICC 6174, saccharomyces cerevisiae (saccharomyces cerevisiae) CICC 1355, Candida utilis (Candida utilis) CICC 31170, Bacillus subtilis (Bacillus subtilis) CICC 24434 and Bacillus licheniformis (Bacillus licheniformis) CGMCC1.10257 respectively.
3. The method for preparing a microbial preparation for regulating the intestinal flora of live pigs according to claim 1, wherein the preparation method comprises the following steps:
the fermentation medium in the step (3) comprises the following components in percentage by mass:
5% of industrial waste molasses, 0.5% of yeast powder, 1% of peptone, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of weakly alkaline small molecular reducing water which is supplemented to 100%.
4. The method for preparing a microbial preparation for regulating the intestinal flora of live pigs according to claim 1, wherein the preparation method comprises the following steps:
the fermentation medium in the step (4) comprises the following components in percentage by mass:
5-8% of industrial waste molasses, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and a weak alkaline small molecule reducing water for supplementing to 100%.
5. A microbial preparation for regulating the intestinal flora of live pigs, which is prepared by the preparation method of any one of claims 1 to 4.
6. The use of the microbial preparation for modulating the intestinal flora of live pigs according to claim 5 in the preparation of a pig feed additive.
7. The pig fermentation feed is characterized by being prepared by fermenting the following raw materials in parts by mass:
8. the method for preparing fermented feed for pigs according to claim 7, characterized by comprising the steps of:
respectively crushing, screening and uniformly mixing corn, bean pulp, wheat bran, broussonetia papyrifera leaves and tea leaves to obtain a mixed raw material; uniformly mixing a microbial preparation for regulating the intestinal flora of live pigs and industrial waste molasses with 50 parts by mass of water, and spraying the mixture onto mixed raw materials; and (3) sealing and fermenting, wherein fermenting for 10-15 days in summer and fermenting for 15-20 days in winter to obtain the pig fermentation material.
9. Use of the fermented feed of claim 7 for preparing a pig feed.
10. The use of the fermented feed for pigs according to claim 9 for preparing a pig feed, characterized in that:
the pig feed contains 5-15 wt% of pig fermentation material.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112154859A (en) * | 2020-08-26 | 2021-01-01 | 河北省科学院生物研究所 | Oyster mushroom cultivation material and preparation method thereof |
| CN112375701A (en) * | 2020-11-11 | 2021-02-19 | 重庆美邦农生物技术有限公司 | Microbial preparation for improving intestinal environment of ruminant such as cattle and sheep and preparation method thereof |
| CN112375702A (en) * | 2020-11-11 | 2021-02-19 | 重庆美邦农生物技术有限公司 | Microbial preparation for improving pig intestinal environment and preparation method thereof |
| CN113243450A (en) * | 2021-01-29 | 2021-08-13 | 上海源耀农牧科技有限公司 | Method for improving quality of mushroom bran feed through multi-strain mixed fermentation |
| CN113462620A (en) * | 2021-08-26 | 2021-10-01 | 福建傲农生物科技集团股份有限公司 | Preparation method and application of composite microbial agent for feed |
| CN113773974A (en) * | 2021-10-12 | 2021-12-10 | 南宁市拜欧生物工程有限责任公司 | Feeding yeast and application thereof |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989005849A1 (en) * | 1987-12-23 | 1989-06-29 | Chr. Hansen's Laboratorium A/S | Lactic acid bacteria for use in fermented milk products and veterinary compositions |
| KR20100137940A (en) * | 2009-06-24 | 2010-12-31 | 순천대학교 산학협력단 | Feed supplement and method for making fermented green tea probiotics |
| CN103110033A (en) * | 2013-03-20 | 2013-05-22 | 邹平泰康生物饲料有限公司 | Pig feed additive and preparation method thereof |
| WO2013151361A1 (en) * | 2012-04-05 | 2013-10-10 | 씨제이제일제당(주) | Novel bacillus subtilis |
| CN103436473A (en) * | 2013-08-23 | 2013-12-11 | 福州大用生物应用科技有限公司 | Composite micro-ecological preparation for pig breeding healthcare |
| CN106615814A (en) * | 2016-12-29 | 2017-05-10 | 中粮生物科技（北京）有限公司 | Feed, preparation method thereof and application of feed to increase of feed intake of lactating sows |
-
2020
- 2020-02-14 CN CN202010093360.6A patent/CN111172077A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989005849A1 (en) * | 1987-12-23 | 1989-06-29 | Chr. Hansen's Laboratorium A/S | Lactic acid bacteria for use in fermented milk products and veterinary compositions |
| KR20100137940A (en) * | 2009-06-24 | 2010-12-31 | 순천대학교 산학협력단 | Feed supplement and method for making fermented green tea probiotics |
| WO2013151361A1 (en) * | 2012-04-05 | 2013-10-10 | 씨제이제일제당(주) | Novel bacillus subtilis |
| CN103110033A (en) * | 2013-03-20 | 2013-05-22 | 邹平泰康生物饲料有限公司 | Pig feed additive and preparation method thereof |
| CN103436473A (en) * | 2013-08-23 | 2013-12-11 | 福州大用生物应用科技有限公司 | Composite micro-ecological preparation for pig breeding healthcare |
| CN106615814A (en) * | 2016-12-29 | 2017-05-10 | 中粮生物科技（北京）有限公司 | Feed, preparation method thereof and application of feed to increase of feed intake of lactating sows |
Non-Patent Citations (4)
| Title |
|---|
| LARSEN N 等: "Characterization of Bacillus spp. strains for use as probiotic additives in pig feed", 《LARSEN N》 * |
| 刘土有 等: "茶叶发酵饲料的特色与应用", 《新农村》 * |
| 蔡玉 等: "构树发酵饲料在猪禽养殖中的应用研究进展", 《畜牧兽医杂志》 * |
| 陈丽仙 等: "益生菌剂对仔猪肠道菌群的影响", 《中国畜牧兽医》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112154859A (en) * | 2020-08-26 | 2021-01-01 | 河北省科学院生物研究所 | Oyster mushroom cultivation material and preparation method thereof |
| CN112375701A (en) * | 2020-11-11 | 2021-02-19 | 重庆美邦农生物技术有限公司 | Microbial preparation for improving intestinal environment of ruminant such as cattle and sheep and preparation method thereof |
| CN112375702A (en) * | 2020-11-11 | 2021-02-19 | 重庆美邦农生物技术有限公司 | Microbial preparation for improving pig intestinal environment and preparation method thereof |
| CN113243450A (en) * | 2021-01-29 | 2021-08-13 | 上海源耀农牧科技有限公司 | Method for improving quality of mushroom bran feed through multi-strain mixed fermentation |
| CN113462620A (en) * | 2021-08-26 | 2021-10-01 | 福建傲农生物科技集团股份有限公司 | Preparation method and application of composite microbial agent for feed |
| CN113773974A (en) * | 2021-10-12 | 2021-12-10 | 南宁市拜欧生物工程有限责任公司 | Feeding yeast and application thereof |
| CN115820515A (en) * | 2023-01-03 | 2023-03-21 | 东北农业大学 | Preparation of cheap lactobacillus PTG medium and application of cheap lactobacillus PTG medium in pickled vegetable fermentation |
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